Translation of mRNAs into proteins must increase to sustain the malignant progression of cancers. Increased translation and increased expression of translation machinery genes in cancers has been generally considered to be a consequence of cellular transformation. However, several recent observations challenge this concept. First, over-expression of the rate-limiting translation initiation factor eIF4E can transform multiple cell types. Second, microRNA (miRNA) repression is frequently reduced in cancers. Because miRNAs often repress oncogenes, reduced miRNA repression increases oncogene expression. Third, eIF4E has been implicated in miRNA-mediated repression and robust increases in eIF4F (a complex of eIF4E, eIF4A and eIF4G) inhibit miRNA function. Therefore, up- regulation of eIF4E may not be a passive participant in cellular transformation but may instead play an active role during oncogenesis by increasing general translation and inhibiting miRNA repression. Human melanomas are optimally suited to test the roles of up-regulated eIF4E in oncogenesis. Human melanomas frequently up-regulate eIF4E, down-regulate miRNAs, down-regulate miRNA- associated proteins including Dicer and Argonautes (Agos), and therefore up-regulate miRNA-targeted oncogenes. Additionally, we have access to a collection of 55 melanoma short-term cultures (MSTCs) that have been annotated by SNP, mRNA, and miRNA expression. Moreover, MSTCs are experimentally-tractable, grow readily ex vivo without requiring immortilization, and thus accurately reflect tumor biology and genetics operant in vivo. To define the roles of up-regulated eIF4E in melanomagenesis, we will knockdown or over-express eIF4E in MSTCs and test proliferation rates, cell-cycle progression, escape from replicative senescence, colony formation in soft agar, and invasive activity. We will then determine the effects eIF4E perturbation on general translation, miRNA repression, or both by performing metabolic labeling and miRNA reporter assays. To comprehensively identify all genes affected by eIF4E perturbation, we will perform microarray analysis of MSTCs before and after eIF4E perturbation. To identify miRNA- repressed mRNAs affected by eIF4E perturbation, we will perform microarray analysis of Ago2 immunopreciptiates. Over-expression of eIF4E should increased expression of miRNA-targeted mRNAs but should decrease Ago2 association of those mRNAs. We will functionally test eIF4E- responsive genes by knocking them down in MSTCs over-expressing eIF4E and re-testing cancer- relevant phenotypes. Dissecting the pathways that link up-regulated eIF4E expression with increased translation of untargeted and miRNA-targeted mRNAs during oncogenesis will elucidate fundamental properties of the malignant progression of melanomas and potentially uncover new therapeutic targets. PUBLIC HEALTH RELEVANCE: Translation must be up-regulated to meet the increased metabolic demands of tumor formation. Experiments in this proposal challenge the notion that increased translation is merely a consequence of cellular transformation. These studies will reveal how increased expression of eIF4E affects translation of all mRNAs, including those selectively repressed by microRNAs, to promote oncogenesis.